Sun G , Hu Z , Min Z , Yan X , Guan Z , Su H , Fu Y , Ma X , Chen YG , Zhang MQ , Tao Q , Wu W .

Xla Wt + ctdspl MO (fig.2.b)
Xla Wt + ctdspl MO (fig.2.b)
Xla Wt + ctdspl MO (fig.2.b)
Xla Wt + ctdspl MO (fig.2.b)
Xla Wt + ctdspl MO (fig.2.b)
Xla Wt + ctdspl MO (fig.2.b)
Xla Wt + ctdspl MO (fig.2.b)
Xla Wt + ctdspl MO (fig.2.b)
Xla Wt + ctdspl MO (fig.2.c)
Xla Wt + ctdspl MO (fig.2.c)
Xla Wt + ctdspl MO (fig.2.d)
Xla Wt + ctdspl MO (fig.2.d)
Xla Wt + ctdspl MO (fig.2.d)
Xla Wt + ctdspl MO (fig.2.d)
Xla Wt + ctdspl MO (fig.3.b)

	FIGURE 1. Expression pattern of scp3 in Xenopus. (A-B) At 8-cell stage, total mRNA from animal and vegetal blastomeres was extracted respectively and analyzed by RNA-seq (A) or qPCR (B). The numbers in (A) indicate the RPKM (reads per kilobase per million mapped reads) of sequencing in two independent experiments (54). Vegetal enriched transcripts bicc was used as a control. Asterisk indicates significantly different (A, FDR<0.05; B, P-value<0.05) between animal and vegetal samples. (C) At indicated stages, RNA from animal and vegetal cells was extracted respectively and the relative mRNA abundance of scp3 was analyzed by qPCR. (D) qPCR shows the temporal expression pattern of scp3. (E) Expression profile of scp3 verified by in situ hybridization. The embryos at 1-cell to stage 10.5 were shown in lateral view. Stage 17 embryo was shown in dorsal view with the anterior end towards the left side. Stage 28 embryo was shown in lateral view with its head towards the left. The arrow indicates the dorsal lip.
	FIGURE 2. SCP3 is essential for Xenopus embryogenesis. (A) MO test in Xenopus embryos. Two-cell stage embryos were coinjected with 40 ng of std MO or scp3 MOs and mRNA coding for Xenopus laevis SCP3-FLAG with indicated doses. At stage 10.5 the embryos were harvested for Western blotting analysis. (B) Standard MO (std MO) or scp3 MOs was injected into 1-cell stage embryos with indicated doses. (C) Two-cell stage embryos were injected with 40 ng of std MO or scp3 MO-1 with or without 750 pg (left) or 200 pg (right) of Xenopus laevis scp3 mRNA as indicated. (D) Uninjected oocytes and scp3 MO-1 injected oocytes were processed by the host-transfer technique and allowed to develop until indicated stages.
	FIGURE 3. SCP3 is required for the expression of Nodal/Activin and BMP target genes during early Xenopus embryogenesis. (A) Oocytes were injected with 20 or 40 ng of scp3 MO-1. After the maturation, host-transfer and fertilization, the embryos were collected at stage 9.5, 10 and 10.5 for qPCR. NC, negative control, uninjected embryos treated the same as injected ones. (B) Two-cell stage embryos were injected with 60 ng of std MO or scp3 MO-1, and analyzed at stage 10.5 using in situ hybridization for the indicated genes.
	FIGURE 4. SCP3 is required for the Nodal/Activin and BMP signals. (A) Reporter assays with ARE and BRE. Two-cell stage embryos were injected in the equatorial region with the ARE or BRE luciferase reporter constructs (20 pg), the Renilla luciferase reporter construct (2 pg), std MO or scp3 MO-1 (60 ng) and indicated mRNAs: Xnr1 (100 pg), bmp4 (1 ng). Embryos were harvested at stage 10.5 and assayed for luciferase activity. (B) Two-cell stage embryos were injected in the animal pole with std MO or scp3 MO-1 (60 ng). Animal caps were excised at stage 8.5 and assayed by qPCR for the expression of markers at stage 10.5. For Activin target assay, animal cap explants were treated with 5 ng/ml of Activin A; and for BMP target assay, 2 ng of bmp4 mRNA was coinjected. (C) Two-cell stage embryos were injected in the animal pole with water or 300 pg of scp3 mRNA. Animal caps were excised at stage 8.5, treated with 5 ng/ml of Activin A and assayed by qPCR for the expression of markers at stage 10.5. (D) Two-cell stage embryos were injected in the animal pole with water or 300 pg of scp3 mRNA. Animal caps were excised at stage 8.5 and assayed by qPCR for the expression of markers at indicated stages. WE, whole embryo.
	FIGURE 5. SCP3 potentiates Nodal/Activin and BMP signals by dephosphorylating linker sites of R-Smads in Xenopus early embryos. (A) Two-cell stage embryos were coinjected with 40 ng of std MO or scp3 MO-1 and mRNA coding for Xenopus laevis Smad2 (2 ng). At stage 10.5 the embryos were harvested to measure the expression of indicated marker genes by qPCR. (B) Two-cell stage embryos were coinjected with 40 ng of std MO or scp3 MO-1 and mRNA coding for Xenopus laevis Smad1 (1 ng). At stage 10.5 the embryos were harvested to measure the expression of indicated marker genes by qPCR. (C) Dynamics of Smad1 and Smad2 phosphorylation during the Xenopus embryogenesis. Embryos were harvested at the indicated developmental stages and their lysates were analyzed by Western Blotting using specific antibodies. LP stands for linker phosphorylation and CP stands for C-terminal phosphorylation. Nodal/Activin- or BMP-responsive Smads are indicted in the right-hand side. (D) Animal and vegetal cells were separated and collected respectively at indicated stages and their lysates were analyzed by Western Blotting using specific antibodies. (E) Oocytes were uninjected or injected with 80 ng of scp3 MO-1. After the maturation, host-transfer and fertilization, the embryos were collected at indicated stages and analyzed using specific antibodies. NC, negative control, uninjected embryos treated the same as injected ones. (F) Two-cell stage embryos were coinjected with 60 ng of std MO or scp3 MO-1 and 2 ng of mRNA coding for wild-type Xenopus laevis Smad2 or mutant Smad2 (S245/250/255A). At stage 10 the embryos were harvested to measure the expression of indicated marker genes by qPCR. (G) Two-cell stage embryos were coinjected with 60 ng of std MO or scp3 MO-1 and 1.5 ng of mRNA coding for wild-type Xenopus laevis Smad1 or mutant Smad1 (S205A). At stage 10 the embryos were harvested to measure the expression of indicated marker genes by qPCR.
	FIGURE 6. SCP3 positively regulates Nodal/Activin and BMP signals in Hep3B cells. (A) Hep3B cells were transfected with 50 nM of NC or SCP3 siRNAs, and 40 h later cells were collected for qPCR. (B) Reporter assays in Hep3B cells. The negative control (NC) siRNA or human SCP3 siRNAs (50 nM) were co-transfected with ARE or BRE luciferase reporter constructs, and 12 h later cells were left untreated or treated with Activin A or BMP4 for 24 h and then collected for reporter activity measurement. (C) Cells were transfected with 50 nM of NC or SCP3 siRNAs, and 40 h later cells were treated with Activin A for 4 h. mRNA levels of Activin induced genes were determined by qPCR. (D) Cells were transfected with 50 nM of NC or SCP3 siRNAs, and 40 h later cells were treated with BMP4 for 4 h. mRNA levels of BMP induced genes were determined by qPCR. (E) Cells were transfected with 100 nM of NC or SCP3 siRNA-1 and cultured for 48 h. After treated with Activin A or BMP4 for 1 h, the cells were collected for Western blotting analysis.
	ctdspl (CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like ) gene expression in sagitally bisected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, lateral view, anterior left, dorsal up.
	ctdspl (CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior left.
	ctdspl (CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.

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